The long-term goal of the proposed research is to expand our understanding of protein synthesis in eucaryotes. Mutations in transfer RNA genes in the eucaryote Saccharomyces cerevisiae will be obtained by isolating nonsense suppressors. The efficiencies and amino acid insertions specified by the suppressors will be determined with the defined iso-1-cytochrome c system. These studies will establish the number of redundant genes coding for particular tRNA species, and may be important in clarifying the reasons for the tRNA redundancy observed in eucaryotes. Genetic mapping studies will be undertaken to see if any of the tRNA genes identified here are tightly linked as they are in the procaryote Escherichia coli. Since Saccharomyces cerevisiae is extensively used in numerous genetic and molecular biology investigations, the additional sophistication brought by isolating numerous nonsense suppressors and identifying as many tRNA genes as possible will be of considerable value. Additional genes involved in protein synthesis will be obtained by selecting for "antisuppressor" mutations which reduce the efficiency of nonsense suppressors. The four groups of antisuppressors that will be isolated are those which act on respectively a tyrosine inserting UAA suppressor, a tyrosine inserting UAG suppressor, a leucine inserting UAG suppressor and an "omnipotent" suppressor. The antisuppressors will be placed into allelic groups and a representative antisuppressor of each locus will be tested for its action on the other suppressors. The mutants isolated may include alterations in tRNA maturation or modification enzymes, ribosomal proteins, or termination factors. A preliminary biochemical characterization of these mutants will be undertaken.